Methods and systems for nucleic acid sequencing by tunneling recognition

ABSTRACT

Embodiments may include a method of analyzing a nucleic acid molecule. The method may include attaching the nucleic acid molecule to a protein. The protein may be attached to a particle with a first diameter. The method may also include applying an electric field to move a first portion of the nucleic acid molecule into an aperture. The aperture may be defined by a first electrode, an insulator, and a second electrode. The aperture may have a second diameter less than the first diameter. The method may further include contacting the first portion of the nucleic acid molecule to both the first electrode and the second electrode. The method may include applying a voltage across the first electrode and the second electrode. The current through the electrodes and the portion of the nucleic acid molecule may be measured, and a nucleotide of the nucleic acid molecule may be identified.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/343,715, filed May 31, 2016, which is incorporated herein byreference in its entirety for all purposes.

BACKGROUND

Nanopore-containing devices have been targeted for the development ofnew analytical methods. Nanopores may have the ability to detect singlemolecules, which may be promising technology in the field of chemicaland biological detection. For example, nanopores may be used fordiagnostics and nucleic acid sequencing. Solid state nanoporebio-sensing may be a rapid single molecule sensing technique. In somecases, solid state nanopores form a channel in an ionic liquid betweentwo electrodes. The two electrodes may not be part of the nanoporeitself but may be positioned in the ionic liquid. As a molecule passesthough the nanopore channel, the current and other electricalcharacteristics through the channel change. These electricalcharacteristics can provide information on the molecule, but fabricationissues may make identifying individual nucleotides in a nucleic acidmolecule difficult. Multiple nucleotides may be present in the apertureat the same time or may pass through the aperture quickly, which may notprovide a strong enough signal for differentiating nucleotides.

Nanopore devices may be used with tunneling recognition. Tunnelingrecognition is based on placing a chemical entity between electrodes,which may be in the nanopore device itself. The orbitals of the chemicalentity will allow electrons to transfer from one electrode to the other,creating a tunneling current. Dimensions and other properties of solidstate nanopores may be difficult to adapt to a mass production process.To sequence nucleic acid molecules with ionic current, nanoporedimensions may need to be on the order of nanometers, which may be lessthan 2 nm. Creating a channel of this size may require precise andexpensive techniques. However, reducing dimensions of the nanopore mayresult in incomplete or poor wetting needed for the nanopore to functionas a sensing device. Improvements in the design and manufacturability ofnanopore-containing devices used in chemical and biological detectionand processes involving the devices are still needed. Design andmanufacturability improvements should not come at the expense ofaccurate and precise analysis. These and other issues are addressed bythe technology described in this document.

BRIEF SUMMARY

Embodiments of the present technology may allow for the sequencing ofnucleic acid molecules by tunneling recognition. A nucleic acid moleculemay be driven into a channel by an electric field or a pressuregradient. In the channel, a portion of the nucleic acid molecule mayenter a gap between two electrodes. Electrodes with a small gap betweenthem may allow for tunneling recognition, but nucleic acid molecules maynot diffuse frequently enough into small gaps to obtain an adequatetunneling signal. The electric field or pressure gradient may drivenucleic acid molecules into the electrode gap more easily andfrequently. Small gaps, which may be 1-2 nm wide, may be difficult orexpensive to manufacture. In some instances, the gap may be between twoelectrodes aligned parallel to each other but not in the same plane,which may be easier to manufacture than a gap between the ends of twocoplanar electrodes. The nucleic acid molecule may be tethered to amotor protein, which may be attached to a particle or a bead. Theparticle may be larger than the channel. The particle may anchor one endof the nucleic acid molecule while an electric field or a flow pulls thenucleic acid molecule through the channel. In this manner, the nucleicacid molecule may be stretched along the channel, and may oscillate inthe channel as a result of Brownian motion. The nucleic acid moleculemay stochastically interact with the tunneling electrodes to yield atunneling signal.

A voltage may be applied to the electrodes. When a portion of thenucleic acid molecule is within the gap between the electrodes and thenucleic acid molecule bridges two electrodes, electrons may tunnelthrough the nucleic acid molecule from one electrode to the other,generating a current. The current may be measured. The nucleic acidmolecule may oscillate in the gap, and the measured current may have anamplitude and frequency. The amplitude and frequency may be variable.The characteristics of the current may aid in identifying a particularnucleotide or base present in the nucleic acid molecule. The electricalcharacteristics may serve almost as a fingerprint in identifying anucleotide of the nucleic acid molecule. The motor protein may feedthrough or pull in the nucleic acid molecule so that a differentnucleotide is in the electrode gap and a different current signal can bemeasured. As a result, an additional nucleotide can be identified. Morenucleotides can be moved into the electrode gap so multiple nucleotidesof the nucleic acid may be identified and the nucleic acid may besequenced.

Embodiments may include a method of analyzing a nucleic acid molecule.The method may include attaching a nucleic acid molecule to a protein.The protein may be attached to a particle with a first diameter. Themethod may also include applying an electric field to move a firstportion of the nucleic acid molecule into an aperture. The aperture maybe defined by a first electrode, a first insulator, and a secondelectrode. The aperture may have a second diameter less than the firstdiameter. The method may further include contacting the first portion ofthe nucleic acid molecule to both the first electrode and the secondelectrode. In addition, the method may include applying a voltage acrossthe first electrode and the second electrode. The current through thefirst electrode, the nucleic acid molecule, and the second electrode maybe measured. Based on the current, a nucleotide of the nucleic acidmolecule may be identified.

Some embodiments may include a nucleic acid molecule analysis system.The system may include a nucleic acid molecule attached to a protein.The protein may be attached to a particle with a diameter. The systemmay also include an aperture defined by a first electrode, a firstinsulator, and a second electrode. The aperture may have a diameter lessthan the diameter of the particle. The system may further include afirst power supply in electrical communication with the first electrodeand the second electrode. In addition, the system may include a secondpower supply configured to apply an electric field through the aperture.

Additional embodiments may include a method of analyzing a nucleic acidmolecule. The method may include attaching a nucleic acid molecule to aprotein. The protein may be attached to a particle with a firstdiameter. The method may also include moving a first portion of thenucleic acid molecule into an aperture defined by an insulator. Theaperture may have a second diameter less than the first diameter. Themethod may further include moving a second portion of the nucleic acidmolecule into a gap between a first electrode and a second electrode.The gap may include a line representing the shortest distance betweenthe electrodes. In addition, the method may include contacting thenucleic acid molecule to both the first electrode and the secondelectrode. In addition, a current through the first electrode, thenucleic acid molecule, and the second electrode may be measured. Basedon the current, a nucleotide of the nucleic acid molecule may beidentified.

Embodiments may include a nucleic acid analysis system. The system mayinclude a nucleic acid molecule attached to a protein. The protein maybe attached to a particle with a first diameter. The system may alsoinclude an aperture defined by a first insulator. The aperture may havea second diameter less than the first diameter. The aperture may have alongitudinal axis perpendicular to the diameter. The system may furtherinclude a first electrode. A portion of the first electrode may extendinto the aperture. The system may also include a second electrode, witha portion of the second electrode extending into the aperture. A planethat includes the portion of the first electrode and the portion of thesecond electrode may be orthogonal to the longitudinal axis. Inaddition, the system may include a first power supply in electricalcommunication with the first electrode and the second electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a side view of a nucleic acid molecule analysis system 100according to embodiments of the present invention.

FIG. 1B shows a top view of a nucleic acid molecule analysis system 100according to embodiments of the present invention.

FIG. 2 is a flowchart of a method 200 of analyzing a nucleic acidmolecule according to embodiments of the present invention.

FIG. 3A shows a nucleic acid molecule contacting electrodes to allow fortunneling recognition in a nucleic acid molecule analysis systemaccording to embodiments of the present invention.

FIG. 3B shows a nucleic acid molecule not contacting electrodes in anucleic acid molecule analysis system according to embodiments of thepresent invention.

FIG. 4A shows a nucleic acid molecule contacting two electrodes beforebeing moved by a molecular motor protein according to embodiments of thepresent invention.

FIG. 4B shows a nucleic acid molecule contacting two electrodes afterbeing moved by a molecular motor protein according to embodiments of thepresent invention.

FIG. 5A shows current characteristics of different nucleic acidmolecules in free flow according to embodiments of the presentinvention.

FIG. 5B shows current characteristics of different nucleic acidmolecules when tethered to a bead according to embodiments of thepresent invention.

FIG. 6A shows a side cut view of a nucleic acid molecule analysis system600 according to embodiments of the present invention.

FIG. 6B shows a top view of a nucleic acid molecule analysis system 600according to embodiments of the present invention.

FIG. 7 shows a flowchart a method 700 of analyzing a nucleic acidmolecule according to embodiments of the present invention.

FIG. 8A shows a nucleic acid molecule contacting two electrodes in anucleic acid molecule analysis system according to embodiments of thepresent invention.

FIG. 8B shows a nucleic acid molecule not contacting two electrodes in anucleic acid molecule analysis system according to embodiments of thepresent invention.

DEFINITIONS

The term “contacting” may refer to bringing one object in proximity toanother object such that electrons may tunnel from one object throughthe other object. At a subatomic level, two objects may never physicallytouch each other as repulsive forces from electron clouds in the objectsmay prevent the objects from coming into closer proximity.

The term “motor protein” may be a molecule that may move a nucleic acidmolecule relative to the molecule. For example, the motor protein mayremain stationary and the nucleic acid molecule may move, or the nucleicacid molecule may remain stationary and the motor protein may move. Amotor protein may include polymerases, helicases, ribosomes,exonucleases, along with other enzymes.

“Nucleic acid” may refer to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The term mayencompass nucleic acids containing known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Examples of such analogs mayinclude, without limitation, phosphorothioates, phosphoramidites, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The term “nucleotide,” in addition to referring to the naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, may beunderstood to refer to related structural variants thereof, includingderivatives and analogs, that are functionally equivalent with respectto the particular context in which the nucleotide is being used (e.g.,hybridization to a complementary base), unless the context clearlyindicates otherwise.

The term “oscillate” may refer to the motion of an object in a fluid asa result of Brownian motion or other forces. An object may oscillatewithout active intervention by a person or a machine. In some cases, anobject may oscillate as a result of an applied electric field or apressure-driven flow.

The term “weak force” may refer to one of the fundamental forces inphysics.

DETAILED DESCRIPTION

Conventional nanopore-based devices currently on the market may containprotein nanopores inserted in metastable lipid bilayers. The lipidbilayers may be fragile and may undermine the stability of the devices.Solid-state atomic scale nanopore layers may be less fragile thanprotein nanopores and have the potential for improved manufacturability.Possible methods involving these devices include confining nucleic acidmolecules in a gap of 2 nm or less between electrodes and recognizingnucleotides and nucleotide sequences using electrons that tunnel throughthe electrodes and the nucleic acid molecule. Solid-state nanopores nearthis dimension may be formed by focusing a beam of electrons or ions ona thin material. This method is difficult to adapt to mass production ofnanopores, nanopore-containing devices, and analytical instruments.Additionally, a nucleic acid molecule may be difficult to force intosuch a small gap between electrodes. Wider channels may allow for anucleic acid molecule to more easily diffuse into a nanopore channel.However, the nucleic acid molecule may pass through a wider channel tooquickly or without the nucleic acid molecule contacting tunnelingelectrodes. As a result, little or no tunneling current may be measured,and the nucleic acid molecule may not be easily sequenced by the device.

Tunneling recognition may be done in geometries that do not necessarilyinclude nanopores. For example, tunneling electrons may be used inscanning tunneling microscopy (STM), which has been used to imageindividual atoms. In theory, STM may be used as well to read a DNAsequence, but STM may rely on a mobile electrode probe, and moving theprobe electrode may move a DNA sample, making obtaining a qualitytunneling signal difficult. Fixing the DNA sample and preventing the DNAfrom moving may improve tunneling recognition.

Devices and methods described herein may allow for a nucleic acidmolecule to be sequenced without forcing the nucleic acid molecule intoa nanopore channel with a diameter of 2 nm or less. Instead, a nanoporechannel may be 10 nm or more in diameter. A nucleic acid molecule mayenter the nanopore channel more easily. Electrode gaps may be 2 nm orless, while the nanopore channel may be a large diameter. For example,electrodes may be stacked on top of each other as layers with aresistive layer in between the two electrodes. The resistive layer mayhave a thickness on the order of 2 nm. The electrodes and the resistivelayer may be exposed in a nanopore channel, with the distance betweenthe electrodes not dependent upon the diameter of the nanopore channel.In some other embodiments, a nanowire may reside in a nanopore channelwith a diameter of 10 nm or more. The nanowire may be separated to forma gap of 2 nm or less, and the two separate parts of the nanowire mayserve as electrodes. In this manner, the gap between electrodes may notbe dependent upon the diameter of the nanopore channel.

In some embodiments, a nucleic acid molecule may be attached to at leastone of a bead and a molecular motor protein. The bead may in at leastone dimension be larger than a dimension of the nanopore channel so thatthe bead may not easily or quickly pass through the nanopore channel.The bead may anchor one end of the nucleic acid molecule in the nanoporechannel, which may allow the nucleic acid molecule to oscillate andrandomly contact electrodes to yield a tunneling signal. The nucleicacid molecule may be driven by an electric field or a pressure-drivenflow, and the bead may reduce the possibility of the nucleic acidmolecule bunching up. The molecular motor protein may feed in (or pullout) the nucleic acid molecule to the channel, allow for additionalportions of the nucleic acid molecule to yield a tunneling signal andtherefore be sequenced.

In some embodiments, nanopore devices and methods may allow for abiological polymer molecule, not just nucleic acid molecules, to beanalyzed. For example, a protein may be analyzed to determine the aminoacids in the protein. The nanopore lengths and diameters may be adjustedfor the size of an amino acid instead of the size of a nucleic acidmolecule. For example, the distance between electrodes may be on theorder of 1-2 nm. In some examples, the distance between electrodes maybe from 0.9 to 2.5 nm. Nanopore devices and methods of tunnelingrecognition are discussed further below.

I. Electrode Stack Tunneling Recognition

Tunneling recognition may be carried out in an electrode stack orsandwich. Two electrodes may be in layers and sandwich an insulator. Thedistance between the electrodes, which may also be the thickness of theinsulator, may be on the order of the size of a nucleotide within anucleic acid, or about 2 nm. The distance between the electrodes may beindependent of any aperture diameter, which may allow for easiermanufacturability. Embodiments described herein may also be modified toanalyze biological polymer molecules other than nucleic acid molecules,including proteins.

A. System

As shown in FIG. 1A and FIG. 1B, some embodiments may include a nucleicacid molecule analysis system 100. FIG. 1A shows a side view of system100, and FIG. 1B shows a top view of system 100. “Side” and “top” areused to describe system 100 in the figures, but system 100 may berotated so that any part of system 100 may point in any direction.System 100 may include a protein 104 attached to a particle 106 having adiameter. If particle 106 is not spherical, particle 106 may have acharacteristic dimension. Particle 106 may have a diameter orcharacteristic dimension in a range from 10 nm to 15 nm, 15 nm to 20 nm,20 nm to 30 nm, 30 nm to 40 nm, 40 nm to 50 nm, or 50 nm or more inembodiments. The particle may be a bead. Protein 104 may be a molecularmotor protein. A molecular motor protein may include polymerases,helicases, ribosomes, and exonucleases.

Protein 104 may be covalently attached to particle 106. For example, thesurface of particle 106 may be modified with Streptavidin, and protein104 may be modified with biotin. In some embodiments, the surface ofparticle 106 may be modified with biotin, and protein 104 may bemodified with biotin. Streptavidin can bind to biotin, which may thenbind protein 104 to particle 106. Similarly, in other examples, thesurface of particle 106 may be modified with SpyTag peptide, and protein104 may be modified with SpyCatcher protein. Alternatively, the surfaceof particle 106 may be modified with SpyCatcher, and protein 104 may bemodified with SpyTag. Covalent bonding between SpyTag and SpyCatcher maybind protein 104 to particle 106. (See B. Zakeri et al., “Peptide tagforming a rapid covalent bond to a protein, through engineering abacterial adhesin,” Proc. Natl. Acad. Sci., 109 (2012), pp. E690-97, thecontents of which are incorporated herein by reference for allpurposes.)

A nucleic acid molecule 102 may be introduced to the system with protein104 attached to particle 106. Nucleic acid molecule 102 may includesingle-stranded DNA or RNA. Nucleic acid molecule 102 may attach toprotein 104.

System 100 may also include an aperture 108 defined by a first electrode110, a first insulator 112, and a second electrode 114. First electrodeand second electrode may include palladium, platinum, gold, or anothernoble metal. At least one of the first electrode and the secondelectrode may be coated with an adapter molecule. The adapter moleculemay undergo weak force chemical interactions with the nucleic acidmolecule. As a result, the adapter molecule may aid the nucleic acidmolecule in bridging or contacting the two electrodes. An adaptermolecule may be organic or organometallic.

First insulator 112 may include a photoresist. The thickness of firstinsulator 112 (i.e., the distance separating first electrode 110 andsecond electrode 114) may be 1 nm or less, 2 nm or less, 3 nm or less, 4nm or less, or 5 nm or less in embodiments. The distance betweenelectrodes may be from 0.9 nm to 2.5 nm, including from 0.9 nm to 1.0nm, 1.0 nm to 1.5 nm, 1.5 nm to 2.0 nm, or 2.0 nm to 2.5 nm. Aperture108 may be defined by a sidewall. The sidewall may include a secondinsulator 118, first electrode 110, first insulator 112, secondelectrode 114, and third insulator 120. Second insulator 118 and thirdinsulator 120 may include a photoresist, glass, and/or silicon nitride.As shown in FIG. 1A, first electrode 110 may be between second insulator118 and first insulator 112. Second electrode 114 may be between firstinsulator 112 and third insulator 120. System 100 may include a sandwichof layers in the following order: second insulator 118, first electrode110, first insulator 112, second electrode 114, and third insulator 120.The layers in the sandwich may be in contact with a neighboring layer orneighboring layers.

Aperture 108 may have a diameter less than the diameter of particle 106.If aperture 108 is not circular or cylindrical, aperture 108 may have acharacteristic dimension less than the characteristic dimension ofparticle 106. The aperture may include a beveled opening or includegeometries similar to a cone. If particle 106 is not circular, particle106 may be described by a characteristic dimension. The characteristicdimension may describe a length, a width, a length of a major axis, or alength of a minor axis. The characteristic dimension may be equal to thecalculated diameter of a sphere having a volume equal to the particle.The diameter or the characteristic dimension of the aperture may be in arange from 10 nm to 15 nm, 15 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40nm, or 40 nm to 50 nm in embodiments. The diameter or the characteristicdimension of aperture 108 may be more than 1 nm, more than 5 nm, or morethan 10 nm shorter than the diameter or characteristic dimension ofparticle 106. The diameter or characteristic dimension of particle 106may be a value that is large enough to prevent particle 106 fromtraversing too quickly through aperture 108 with a diameter orcharacteristic dimension.

System 100 may further include a first power supply 116 in electricalcommunication with first electrode 110 and second electrode 114. Firstpower supply 116 may apply a voltage to first electrode 110 and secondelectrode 114. First power supply 116 may be configured to maintain adesired current or a desired voltage. The current passing through bothelectrodes may be measured by a meter device 122. The current passingthrough both electrodes may include current that tunnels through nucleicacid molecule 102. Meter device 122 may include a current meter, anoscilloscope, or other current-measuring meters. In addition, system 100may include a second power supply 124 configured to apply an electricfield through aperture 108. This electric field may drive nucleic acidmolecule 102 through aperture 108. A liquid may be disposed in aperture108. The liquid may be an electrically conductive solution and allownucleic acid molecule 102 to flow through aperture 108. Second powersupply 124 may be in electrical communication with a third electrode 126and a fourth electrode 128. Third electrode 126 and fourth electrode 128may be disposed in a liquid and disposed on opposite ends of aperture108.

System 100 may be one of a plurality of analysis systems, allowing forthe analysis of thousands to millions of nucleic acid molecules. Aplurality of analysis systems may share the same second power supplyused to drive nucleic acid molecules through the aperture. In the caseof a plurality of analysis systems, the diameters and characteristicdimensions may be the mean or median diameter or characteristicdimension of the plurality. Using a plurality of analysis systems mayallow multiplexing, which may be an advantage over other tunnelingrecognition systems.

Systems may also include a biological polymer analysis system, where abiological polymer molecule, instead of a nucleic acid molecule, isanalyzed. For example, a system similar to system 100 may be used toanalyze a protein and the amino acids of the protein.

B. Method

As shown in FIG. 2, embodiments may include a method 200 of analyzing anucleic acid molecule.

At block 202, method 200 may include attaching a nucleic acid moleculeto a protein. The protein typically may have a binding site for anucleic acid, similar to a binding site of an enzyme for a substrate.The protein and nucleic acid may form a non-covalent bond, with astrength depending on the protein or enzyme involved. The protein may beattached to a particle with a first diameter. The nucleic acid molecule,protein, and particle may be any described herein.

At block 204, method 200 may also include applying an electric field tomove a first portion of the nucleic acid molecule into an aperture. Thefirst portion of the nucleic acid molecule may include a nucleotide. Theaperture may be defined by a first electrode, a first insulator, and asecond electrode. The aperture may have a second diameter less than thefirst diameter. The nucleic acid molecule may be in a fluid, and theelectric field may be applied through electrodes in the fluid, where theelectrodes may be located on opposite ends of the aperture.

At block 206, method 200 may further include contacting the firstportion of the nucleic acid molecule to both the first electrode and thesecond electrode (block 206).

FIG. 3A shows a portion of nucleic acid molecule 302 contacting firstelectrode 304 and second electrode 306 across insulator 308. When aportion of nucleic acid molecule 302 contacts both electrodes, themolecule may also be considered to bridge both electrodes. When aportion of nucleic acid molecule 302 contacts both electrodes, electronsmay tunnel from one electrode to the other. The current generated by thetunneling electrodes may be measured. As shown in FIG. 3B, if nucleicacid molecule 302 does not contact first electrode 304 or secondelectrode 306, then electrons may not tunnel through the electrodes andno current may be measured.

At block 208, method 200 may include applying a voltage across the firstelectrode and the second electrode. The electrodes may be consideredtunneling electrodes and may be any electrode described herein. Thevoltage may be direct current or alternating current voltage. Thevoltage may be applied in pulses or in a waveform (e.g., sine, square,triangle, or sawtooth). Method 200 may also include applying a currentthrough the first electrode and the second electrode.

At block 210, method 200 may include moving the nucleic acid moleculesuch that a second portion of the nucleic acid molecule contacts boththe first electrode and the second electrode. The second portion of thenucleic acid molecule may be in a different location than the firstportion of the nucleic acid molecule. The second portion of the nucleicacid molecule may include a nucleotide. The nucleic acid molecule may bemoved using the protein. In some cases, the second portion of thenucleic acid molecule may be positioned closer to the protein than thefirst portion of the nucleic acid molecule is positioned to the protein.In other cases, the second portion of the nucleic acid molecule may bepositioned farther from the protein than the first portion is positionfrom the protein.

FIG. 4A and FIG. 4B show diagrams of how a nucleic acid molecule 402 maybe moved by a protein 404. In FIG. 4A, nucleic acid molecule 402contacts a first electrode 406 and a second electrode 408 acrossinsulator 410. Nucleic acid molecule 402 may contact the electrodes in away such that electrons tunnel through a nucleotide 412. Nucleotide 412may be any of the nucleotide bases, including A, C, G, T, and U, and maybe a labeled, artificial, or methylated nucleotide. A nucleotide 414 maybe closer to protein 404 than nucleotide 412 is to protein 404, while anucleotide 416 may be farther away. Electrons may not tunnel througheither nucleotide 414 or 416 because these nucleotides are notpositioned between the electrodes. Protein 404 may move nucleic acidmolecule 402 away from or toward a particle 420. FIG. 4B shows anexample of nucleic acid molecule 402 from FIG. 4A moving away fromparticle 420. Protein 404 may move nucleic acid molecule 402 so thatnucleotide 412 is no longer aligned so that electrons may tunnel throughnucleotide 412. Instead, nucleotide 414 may be aligned so that electronstunnel through nucleotide 414. Electrons tunneling through a differentnucleotide may generate different current characteristics. In someembodiments, electrons may tunnel through two or more nucleotides.

The nucleic acid molecule may oscillate in the aperture. The nucleicacid molecule may oscillate as a result of Brownian motion. In someembodiments, oscillation may be partly or wholly a result of an appliedelectric field, pressure-driven flow, or a change in an electric fieldor flow. The oscillation of the nucleic acid molecule may change theportion of the nucleic acid molecule contacting or bridging theelectrodes. In some instances, the nucleic acid molecule may oscillatebetween a configuration similar to FIG. 3A, where a portion of thenucleic acid molecule contacts both electrodes, and a configurationsimilar to FIG. 3B, where a nucleic acid molecule does not contact bothelectrodes. In some configurations, a portion of the nucleic acidmolecule may still make contact with both electrodes but in a mannerthat allows the electrons to more easily or less easily tunnel through.For example, the nucleic acid molecule may not make a full contact withone of the electrodes. As a result, the oscillation may affect thecurrent passing through the electrodes and the nucleic acid.

At block 212, the current through the first electrode, the first portionof the nucleic acid molecule, and the second electrode may be measuredwhile the nucleic acid molecule oscillates in the aperture. Theoscillating nucleic acid may affect at least one of the amplitude, pulsewidth, and frequency of the current. The pulse width may also beconsidered a dwell time, which may be related to the duration of acertain portion of the nucleic acid molecule remaining in contact withboth electrodes. In some embodiments, voltage or resistance may bemeasured instead of or in addition to current. In some embodiments, theelectrical characteristic may be transformed by a mathematicaloperation, such as a Fourier transform, and the transform may beanalyzed. In embodiments, the measured electrical characteristic may becompared to the applied voltage and adjusted to isolate the oscillationof the nucleic acid molecule.

At block 214, based on the current, the nucleotide of the first portionof the nucleic acid molecule may be identified. The current may becompared to a calibration current measured from a known nucleotide or aknown sequence. Nucleotides or sequences may have a current pattern thatserves as a fingerprint to identify the nucleotide or sequence. Theamplitude, frequency, and/or pulse width of the measured current may beused to identify the unknown nucleotide or sequence based on a knownnucleotide or sequence. The amplitude may depend on the individualnucleotide and the resistance of the nucleotide. The frequency maydepend on how the nucleotide and/or neighboring nucleotides oscillate inthe aperture. For example, a pattern may be recognized and matched to aknown nucleotide or sequence. An exact match may not be needed. Instead,if a current measured from an unknown nucleotide or sequence has acertain threshold level of amplitude, frequency, and/or pulse width, thenucleotide or sequence may be identified. Analyzing the currentcharacteristics may be similar to analyzing electrical characteristicsfrom resonant-tunneling diodes. Tunneling recognition of nucleotides maybe similar to tunneling recognition of amino acids as described by Zhaoet al., “Single-molecule spectroscopy of amino acids and peptides byrecognition tunneling,” Nature Nanotech. 9, (2014) 466-73, the contentsof which are incorporated herein by reference for all purposes.

In embodiments, one or more nucleotides may be labeled. A nucleotide maybe labeled with a chemical compound that yields tunneling currents thatare stronger than the nucleotide without the chemical compound. Thelabel may be specific to the type of nucleotide it is attached. Labelsmay include conjugated organic molecules, organometallic compounds, ormetal clusters. The labeled nucleotides may be included as labelednucleotide tri-phosphates in the analysis system.

In some embodiments, the nucleic acid molecule may be circular DNA or aDNA formed with repeated sequences. In embodiments with circular DNA,protein 104 may be a polymerase. The polymerase may copy the circularDNA, and as the polymerase accomplishes a full circle, thesingle-stranded DNA just synthesized may be moved into the aperture. The“threading” of the aperture by the single-stranded DNA may be controlledby the kinetics of the polymerase and the availability of nucleotidetri-phosphate. As a result of the circular DNA template, thesingle-stranded DNA may include repeated sequences. The repeatedsequences may rotate through the electrodes, which may allow for abetter signal to identify nucleotides.

In other embodiments, methods may be used to analyze a biologicalpolymer other than a nucleic acid molecule. For example, the aminoacids, rather than the nucleotides, of a protein, rather than a nucleicacid molecule, may be analyzed by methods similar to method 200.

C. Example

FIG. 5A and FIG. 5B show how the current characteristics of a nucleicacid molecule 502 that is allowed to flow freely compared with a nucleicacid molecule 504 tethered to a bead 506. Nucleic acid molecule 502 mayfit in a channel defined by glass, palladium electrodes, an insulatinglayer, and silicon nitride, but nucleic acid molecule 502 may flowthrough the channel too quickly. In some cases, nucleic acid molecule502 may reside for some time in the channel but may bunch up rather thanbeing a straight or mostly straight strand. Bunching up may mean thatthe nucleic acid molecule doubles against itself in some areas, whichmay cause the nucleic acid molecule to not bridge the electrodes. Incontrast, nucleic acid molecule 504 tethered to bead 506 may not flowthrough the channel because bead 506 has a diameter larger than thesmallest dimension of the channel opening. In addition, bead 506 mayanchor nucleic acid molecule 504 in the channel while an electric fieldpulls the nucleic acid molecule through the channel. As a result,nucleic acid molecule 504 may be straight or mostly straight and may notbunch up. Even though nucleic acid molecule 504 may not bunch up,Brownian motion may move nucleic acid molecule 504 so that electrons maytunnel through nucleic acid molecule 504.

Both FIG. 5A and FIG. 5B show an aperture with a bevel in the glassinsulator. This bevel may be a result of a the nano-fabrication process.In many cases, the sidewalls of the aperture may not be completelystraight and may be beveled or slanted as would be expected from thecontrol and accuracy of fabrication techniques, including etching. Amolecular motor protein was not used with the embodiment in FIG. 5B tosimplify the experimental setup.

Graphs 508, 510, 512, and 514 show current measurements resulting fromelectrons tunneling through nucleic acid molecules. Graph 508 showscurrent measurements from a nucleic acid molecule, “Oligo A,” that isnot tethered to a bead, while graph 510 shows current measurements fromthe same type of nucleic acid molecule but tethered to a bead. Graph 512shows current measurements from a different nucleic acid molecule,“Oligo C,” that is not tethered to a bead, while graph 514 shows currentmeasurements from Oligo C tethered to a bead. Graphs 508 and 512generally show less time with non-zero current, when compared withcounterpart graphs 510 and 514. In graph 510, for most of the time,current is non-zero, while in graph 508, current is zero or near zerofor most of the time. Additionally, graph 510 shows a much morerepetitive or periodic pattern of current fluctuations compared to graph508. A current between the minimum and maximum current may indicate poorcontact, a more resistive nucleotide, or the nucleotide in the processof forming or undoing contact with the electrodes, as these reasons mayimpact the quality of the tunneling effect.

Additional differences in current characteristics between tethered andnon-tethered nucleic acid molecules can be seen in graphs 512 and 514.In graph 512, current is zero almost all of the time, with the exceptionof a few spikes in current. This pattern indicates that the nucleic acidmolecule is not contacting the electrodes frequently. In contrast, graph514 shows many spikes in current, occurring in a periodic or fairlyperiodic pattern. The pattern in graph 514 indicates that the nucleicacid molecule is contacting the electrodes frequently.

Graphs 508, 510, 512, and 514 show that nucleic acid molecules tetheredto a bead provide stronger current signals than nucleic acid moleculesthat are allowed to flow freely through a channel. Signals may beclearer with tethered nucleic acid molecules and may allow for easierand more accurate identification of nucleotides.

II. Coplanar Electrodes in a Tunneling Break Junction

In some embodiments, tunneling recognition may be carried out betweentwo electrodes that are substantially coplanar. The electrodes may beseparated by a distance on the order of 2 nm, which may allow fortunneling recognition of a nucleotide. The electrodes may extend into anaperture, which has a diameter greater than 2 nm. The distance betweenthe electrodes may then be independent of the aperture diameter.

In some embodiments, nanopore devices and methods may allow for abiological polymer molecule, not just nucleic acid molecules, to beanalyzed. For example, a protein may be analyzed to determine the aminoacids in the protein. The nanopore lengths and diameters may be adjustedfor the size of an amino acid instead of the size of a nucleic acidmolecule. For example, the distance between electrodes may be on theorder of 1-2 nm. Nanopore devices and methods of tunneling recognitionare discussed further below.

A. System

As shown in FIG. 6A and FIG. 6B, embodiments may include a nucleic acidanalysis system 600. FIG. 6A is a side cut view, and FIG. 6B is a topview. The top view may be on a surface of a silicon wafer or amicrofluidic chip. System 600 may include a protein 604 attached to aparticle 606 with a first diameter. Protein 604 may be any proteindescribed herein, and particle 606 may be any particle described herein.

A nucleic acid molecule 602 may be introduced to the system with protein104 already attached to particle 606. Nucleic acid molecule 602 mayinclude any nucleic acid molecule described herein. Nucleic acidmolecule 602 may attach to protein 604.

System 600 may also include an aperture 608 defined by a first insulator610. The first insulator may include Si₃N₄, oxide insulators (e.g.,SiO₂, Al₂O₃, oxide silicates), glass, quartz, or any insulator materialdescribed herein. Aperture 608 may have a second diameter smaller thanthe first diameter. The second diameter of aperture 608 may be largerthan the diameter of the aperture 108 in FIG. 1A and 1B. The seconddiameter may be up to 50 nm. Aperture 608 may have a longitudinal axis612 perpendicular to the diameter.

System 600 may further include a first electrode 614. A portion of firstelectrode 614 may extend into aperture 608. The first electrode mayinclude gold, palladium, platinum, or any electrode material describedherein.

System 600 may also include a second electrode 616, with a portion ofsecond electrode 616 extending into the aperture. A plane that includesthe portion of first electrode 614 and the portion of second electrode616 may be orthogonal to the longitudinal axis. First electrode 614 andsecond electrode 616 may define a gap, which includes the shortestdistance between the two electrodes. The shortest distance between firstelectrode 614 and second electrode 616 may be less than 1 nm, from 1 nmto 2 nm, from 2 nm to 3 nm, from 3 nm to 5 nm, from 5 nm to 10 nm, from0.9 nm to 2.5 nm, from 0.9 nm to 1.0 nm, from 1.0 nm to 1.5 nm, from 1.5nm to 2.0 nm, or from 2.0 nm to 2.5 nm in embodiments.

First electrode 614 and second electrode 616 may be formed by breaking asingle nanowire into the two electrodes. Breaking may involve differenttechniques to separate a nanowire into two pieces that do not contactthe other piece. Breaking the single nanowire may be by physicallybending a semiconductor chip with the nanowire on it. Bending the chipmay then break the wire and may create a gap between the two parts ofthe broken nanowire. The second diameter of aperture 608 may be largeenough to accommodate the mechanical bending of the chip. The electrodemay also be broken by being cut with a beam or by applying a highelectrical potential. The second electrode may be any electrode materialdescribed herein.

FIGS. 6A and 6B show first electrode 614 and second electrode 616 havinga triangular or conical shape in the aperture. The shape of theelectrodes may be formed by repeated back-and-forth bending of the chipuntil the electrodes touch. An applied electrical potential then mayhelp shape the tips of the electrodes. In other embodiments, the portionof first electrode 614 closest to second electrode 616 may not come to apoint. Instead, the ends of the electrodes may be flat or hemispherical.

In addition, system 600 may include a first power supply 618 inelectrical communication with first electrode 614 and the secondelectrode 616. First power supply 618 may be apply a voltage to firstelectrode 614 and second electrode 616. First power supply 618 may beconfigured to maintain a desired current or a desired voltage. Currentpassing through the electrodes may be measured by a meter device 622.Meter device 622 may be configured to measure current through theelectrodes along and through a portion of the nucleic acid moleculebridging the electrodes. Meter device 622 may be any meter devicedescribed herein.

First electrode 614 may be between first insulator 610 and a secondinsulator 620. Similarly, second electrode 616 may be between firstinsulator 610 and second insulator 620. First insulator 610 and secondinsulator 620 may include silicon nitride, silicon dioxide, glass,quartz, and other insulating materials described herein. Photoresist maybe difficult to shape on the scale needed for the first insulator in atunneling break junction. The insulator material may extend farther thanwhat is shown in FIGS. 6A and 6B, for the insulator material may be themajority material of a microfluidic chip.

In some embodiments, system 600 may include a second power supply (notshown). The second power supply may be configured to apply an electricfield that would move a nucleic acid molecule through aperture 608. Theelectric field may be applied across the layers that make up theaperture. As with system 100 in FIG. 1, system 600 may have a liquiddisposed in aperture 608. The power supply may be in electricalcommunication with a third electrode and a fourth electrode. The thirdelectrode and fourth electrode may be disposed in a liquid and disposedon opposite ends of aperture 608.

First insulator 610 and second insulator 620 may be attached to amicrofluidic chip, including a silicon chip. The insulators may bedeposited or formed on top of a semiconductor material or may bepatterned from the semiconductor material. The semiconductor materialmay include a silicon wafer.

Systems may also include a biological polymer analysis system, where abiological polymer molecule, instead of a nucleic acid molecule, isanalyzed. For example, a system similar to system 600 may be used toanalyze a protein and the amino acids of the protein.

B. Method

As shown in FIG. 7, additional embodiments may include a method 700 ofanalyzing a nucleic acid molecule.

At block 702, method 700 may include attaching a nucleic acid moleculeto a protein. The protein may be attached to a particle with a firstdiameter.

At block 704, method 700 may also include moving a portion of thenucleic acid molecule into an aperture defined by an insulator. Theportion of the nucleic acid molecule may include a nucleotide. Theaperture may have a second diameter less than the first diameter.

Moving the portion of the nucleic acid molecule may include applying anelectric field to drive the nucleic acid molecule through the aperture.In these and other embodiments, moving the portion may include movingthe nucleic acid molecule with a pressure-driven liquid flow. Aconvective liquid flow may be used to push the nucleic acid moleculethrough the aperture. A flowing liquid to move nucleic acid moleculesmay be more effective with a system similar to system 600 in FIG. 6A and6B compared to system 100 in FIG. 1A and 1B. System 100 may be onlyabout 10 to 20 nm thick, and a liquid flow may break the sandwich ofvarious layers. On the other hand, system 600 may be fabricated to bethicker than system 100. Additionally, parts of system 600, includingthe insulator, may be strongly attached to a wafer or other material,which may provide greater stability in a flowing liquid.

Method 700 may further include moving the portion of the nucleic acidmolecule into a gap between a first electrode and a second electrode.The gap may include a line representing the shortest distance betweenthe electrodes. Moving the portion of the nucleic acid molecule mayinclude using the same forces that moved the portion into the aperture.

At block 706, method 700 may include contacting the portion of thenucleic acid molecule to both the first electrode and the secondelectrode. The portion of the nucleic acid molecule may contact thefirst electrode and the second electrode within the aperture.

FIG. 8A shows a nucleic acid molecule 802 contacting first electrode 804and second electrode 806, with both electrodes electrically isolated ininsulators 808 and 810. When nucleic acid molecule 802 contacts bothelectrodes, the molecule may also be considered to bridge bothelectrodes. During this contact, electrons may tunnel from one electrodeto the other. The current generated by the tunneling electrodes may bemeasured. As shown in FIG. 8B, if nucleic acid molecule does not contactfirst electrode 804 or second electrode 806, then no electrons maytunnel through the electrodes and no current may be measured. As shownin FIG. 8B, nucleic acid molecule 802 may contact only first electrode804 and not second electrode 806.

At block 708, method 700 may include applying a voltage across the firstelectrode and the second electrode. The electrodes may be consideredtunneling electrodes and may be any electrode described herein. Thevoltage may be direct current or alternating current voltage. Thevoltage may be applied in pulses or in a waveform (e.g., sine, square,triangle, or sawtooth). Method 700 may also include applying a currentthrough the first electrode and the second electrode

At block 710, a current through the first electrode, the nucleic acidmolecule, and the second electrode may be measured while the nucleicacid molecule oscillates in the aperture.

At block 712, based on the current, the nucleotide of the portion of thenucleic acid molecule may be identified, similar to methods describedherein.

In other embodiments, methods may be used to analyze a biologicalpolymer other than a nucleic acid molecule. For example, the aminoacids, rather than the nucleotides, of a protein, rather than a nucleicacid molecule, may be analyzed by methods similar to method 700.

In the preceding description, for the purposes of explanation, numerousdetails have been set forth in order to provide an understanding ofvarious embodiments of the present technology. It will be apparent toone skilled in the art, however, that certain embodiments may bepracticed without some of these details, or with additional details.

Having described several embodiments, it will be recognized by those ofskill in the art that various modifications, alternative constructions,and equivalents may be used without departing from the spirit of theinvention. Additionally, a number of well-known processes and elementshave not been described in order to avoid unnecessarily obscuring thepresent invention. Additionally, details of any specific embodiment maynot always be present in variations of that embodiment or may be addedto other embodiments.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed.The upper and lower limits of these smaller ranges may independently beincluded or excluded in the range, and each range where either, neither,or both limits are included in the smaller ranges is also encompassedwithin the invention, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a method” includes aplurality of such methods and reference to “the particle” includesreference to one or more particles and equivalents thereof known tothose skilled in the art, and so forth. The invention has now beendescribed in detail for the purposes of clarity and understanding.However, it will be appreciated that certain changes and modificationsmay be practice within the scope of the appended claims.

What is claimed is:
 1. A method of analyzing a nucleic acid molecule,the method comprising: attaching the nucleic acid molecule to a proteinand a particle having a first diameter; moving a portion of the nucleicacid molecule into an aperture defined by a first insulator, theaperture having a second diameter less than the first diameter, and theportion comprising a nucleotide; applying a voltage across a firstelectrode and a second electrode; contacting the portion of the nucleicacid molecule to both the first electrode and the second electrodewithin the aperture; measuring a current through the first electrode,the portion of the nucleic acid molecule, and the second electrode; andidentifying the nucleotide of the portion of the nucleic acid moleculebased on the current.
 2. The method of claim 1, wherein the protein is amolecular motor protein selected from the group consisting ofpolymerases, helicases, ribosomes, and exonucleases.
 3. The method ofclaim 1, wherein the nucleic acid molecule comprises single-strandedDNA, RNA, or circular DNA.
 4. The method of claim 1, further comprising:measuring an amplitude and a frequency of the current while the nucleicacid molecule oscillates in the aperture.
 5. The method of claim 1,further comprising: measuring a pulse width of the current while thenucleic acid molecule oscillates in the aperture.
 6. The method of claim1, further comprising comparing the current to a calibration currentmeasured from a known nucleotide.
 7. The method of claim 1, wherein thenucleotide comprises a nucleotide labeled with a chemical compound thatyields an increased tunneling current relative to an unlabelednucleotide.
 8. The method of claim 1, further comprising moving theportion of the nucleic acid molecule into a gap between the firstelectrode and the second electrode, the gap comprising a linerepresenting the shortest distance between the first electrode and thesecond electrode.
 9. The method of claim 8, wherein the shortestdistance between the first electrode and the second electrode is 2 nm orless.
 10. The method of claim 1, wherein moving the portion of thenucleic acid molecule into the aperture comprises moving the portion ofthe nucleic acid molecule with an applied electric field or apressure-driven flow.
 11. The method of claim 1, wherein: the aperturehas a longitudinal axis perpendicular to the second diameter, a portionof the first electrode extends into the aperture, a portion of thesecond electrode extends into the aperture, a plane comprising theportion of the first electrode and the portion of the second electrodeis orthogonal to the longitudinal axis.
 12. The method of claim 1,wherein: the aperture is defined by a sidewall, the sidewall comprises asecond insulator, the first electrode, the first insulator, the secondelectrode, and a third insulator, the second insulator is in contactwith the first electrode, the first electrode is in contact with thefirst insulator, the first insulator is in contact with the secondelectrode, and the second electrode is in contact with the thirdinsulator.
 13. The method of claim 1, wherein: moving the portion of thenucleic acid molecule into the aperture comprises applying an electricfield, and the aperture is further defined by a first electrode and asecond electrode.
 14. The method of claim 13, wherein the seconddiameter is in a range from 10 nm to 15 nm.
 15. The method of claim 13,wherein the first diameter is 10 nm or more.
 16. The method of claim 13,wherein the first insulator has a thickness of 2 nm or less.
 17. Themethod of claim 13, wherein the portion of the nucleic acid molecule isa first portion of the nucleic acid molecule, the method furthercomprising: moving, using the protein, the nucleic acid molecule suchthat a second portion of the nucleic acid molecule contacts both thefirst electrode and the second electrode, wherein: the second portion ofthe nucleic acid molecule is at a different location than the firstportion of the nucleic acid molecule, and the second portion of thenucleic acid molecule comprises a second nucleotide.
 18. A nucleic acidmolecule analysis system, the system comprising: a protein attached to aparticle, the particle having a first diameter; an aperture defined by afirst electrode, a first insulator, and a second electrode, the aperturehaving a second diameter less than the first diameter; a first powersupply in electrical communication with the first electrode and thesecond electrode; a second power supply configured to apply an electricfield through the aperture to move the protein attached to the particleto the aperture; and a meter device configured to measure a currentthrough the first electrode and the second electrode when a firstportion of a nucleic acid molecule attached to the protein contacts thefirst electrode and the second electrode.
 19. The system of claim 18,wherein the system further comprises the nucleic acid molecule attachedto the protein.
 20. The system of claim 18, wherein the first insulatorcomprises a photoresist.
 21. The system of claim 18, wherein: theaperture is further defined by a sidewall, and the sidewall comprises asecond insulator, the first electrode, the first insulator, the secondelectrode, and a third insulator.
 22. The system of claim 21, wherein:the first electrode is disposed between the second insulator and thefirst insulator, and the second electrode is disposed between the firstinsulator and the third insulator.
 23. The system of claim 19, whereinat least one of the first electrode and the second electrode is coatedwith an adapter molecule, wherein the adapter molecule undergoes weakforce chemical interactions with the nucleic acid molecule.
 24. Anucleic acid analysis system, the system comprising: a protein attachedto a particle, the particle having a first diameter; an aperture definedby a first insulator, the aperture having a second diameter smaller thanthe first diameter, and the aperture having a longitudinal axisperpendicular to the second diameter; a first electrode, wherein aportion of the first electrode extends into the aperture; a secondelectrode, wherein a portion of the second electrode extends into theaperture and wherein a plane comprising the portion of the firstelectrode and the portion of the second electrode is orthogonal to thelongitudinal axis; a first power supply in electrical communication withthe first electrode and the second electrode; and a meter deviceconfigured to measure a current through the first electrode and thesecond electrode when a first portion of a nucleic acid moleculeattached to the protein contacts the first electrode and the secondelectrode.
 25. The system of claim 24, further comprising the nucleicacid molecule attached to the protein.
 26. The system of claim 24,further comprising a second power supply configured to apply an electricfield through the aperture.
 27. The system of claim 24, wherein thefirst electrode is disposed between the first insulator and a secondinsulator.
 28. The system of claim 24, wherein the second electrode isdisposed between the first insulator and a second insulator.
 29. Thesystem of claim 24, wherein: the first electrode and the secondelectrode define a gap, the gap comprises the shortest distance betweenthe first electrode and the second electrode, and the shortest distancebetween the first electrode and the second electrode is 2 nm or less.30. The system of claim 29, wherein the system is attached to amicrofluidic silicon chip.